JP2671129B2 - Method of suppressing interleukin-1 release and reducing interleukin-1 mediated symptoms - Google Patents

Description

TECHNICAL FIELD The present invention relates to a composition that suppresses interleukin-1 release and reduces interleukin-1 mediated symptoms.

PRIOR ART Substituted alkylidene dithio-bis-used in the present invention
(Substituted) phenols are described in US Pat. No. 3,576,883,
Compounds of the type disclosed in 3,786,100, 3,862,332, and 3,897,500, which can be made by the methods disclosed in these patents. It is one of the alkylidene dithio-bis- (substituted) phenols
4,4 '-(Isopropylidenedithio) bis (2,6-ditertiarybutyl) phenol is known by the generic name "Probucol" and is used as a hypocholesterolemic drug. Probucol lowers serum cholesterol,
High density riboprotein (HDL) and low density riboprotein (L
It is known to reduce both DL) cholesterol. Probucol has been shown to suppress oxidative alterations in LDL, and this effect was postulated to be likely to suppress tumor formation.
Szewicz) et al., Journal of Lipid Research 25: 1206 (1984); and Parthasarathy.
J. Clin. Invest. 77, 641 (1985)].

Interleukin-1 (IL-1) is the name for a family of molecules with multiple biological effects. The name Interleukin-1 was first proposed in 1979, and earlier literature referred to it by other names. Murphy
Et al., British Journal of Reumatology, 1985, 24 (Appendix 1), pages 6-9, and Oppenheim.
m) et al., Immunology Today, Vol. 2, pp. 45-55 (1986). IL-1 is secreted by stimulated macrophages and has several important biological effects such as mediation of T-type lymphocyte proliferation and febrile and proinflammatory effects.

IL-1 is summarized in the two references above. IL-1 is described as mediating the acute response of inflammation and having fever and proinflammatory effects. IL-1 induces connective tissue changes. It was also demonstrated to induce the release of degrading enzymes from mesenchymal cells present at sites of bone erosion such as rheumatoid arthritis. Billingham, Br
it. J. Rheumatology, 1985, 24 (Appendix 1) 25-28:
See Dayer, Brit. J. Rheumatology, 1985, Volume 24 (Appendix 1), pages 15-20. Acute phase protein production in hepatocytes during the acute phase of inflammation is IL-1 and IL-
It is mediated by other cytokines such as 6. Witcher, Brit. J. Rheumatology, 1985,
See Volume 24 (Appendix 1), pages 21-24.

IL-1 is also involved as a vector for psoriasis of inflammatory skin diseases. Camp et al., J. Immunology, 1986, 1
Volume 37, pages 3469-3474, and Ristow, Proc. Nat
l. Acad. Sci. USA, 1987, 84, pp. 1940-1944. IL-1 is cytotoxic to insulin-producing beta cells in the pancreas and is thus a causative factor in the development of some forms of diabetes mellitus [Bendtzen et al., Sc.
ience, 1986, 232, 1545-1547, and Marx (Mar
x), Science, 1988, 239: 257-258]. Also IL-1
Appears to be involved in the development of atherosclerotic lesions and atherosclerotic plaques [Marx, Science 1988, 239: 257-258]. IL-1 stimulates the growth and proliferation of vascular smooth muscle. This effect is
Greater in the absence or inhibition of endogenous prostaglandins that lead to thickening of the vascular wall, such as that caused by neoplasia [Libby et al., Fed. Proc. 1987 3
Vol. 1, No. 3, p. 975, abstract p. 3837].

[Problems to be Solved by the Invention] It would be advantageous if IL-1 release could be controlled and IL-1 mediated effects could be treated. It would also be advantageous to control or treat IL-1 mediated inflammation without the side effects known to be associated with the use of anti-inflammatory steroids and non-steroidal anti-inflammatory agents at the same time.

[Means for Solving the Problems] In the present invention, the formula [Wherein R is tert-butyl or tert-pentyl, R'and R "are hydrogen, methyl, ethyl, propyl, butyl, or pentyl, but R'and R" are not isopropyl, R ₁ is hydrogen, methyl or ethyl,
R ₂ is lower alkyl having 1 to 6 carbon atoms, or 3-
Is a 6 carbon atom keto-substituted lower alkyl, or R ₂ is of the formula Where n is 2, 3 or 4 and R, R ', R "and R ₁ are as defined above] substituted alkylidene dithio-bis- (substituted)- It has been discovered that phenolics can be used to suppress IL-1 secretion, such compounds inhibiting IL-1 secretion, IL-1.
It can be administered to animals (including humans) for the inhibition or treatment of -1 mediated effects and the inhibition or treatment of IL-1 mediated inflammation. The compounds can be administered to suppress or treat the IL-1-mediated effects in conditions such as inflammation, psoriasis, atherosclerosis and diabetes.

In the compositions of the present invention, an effective amount of one or more of the compounds corresponding to the above formulas is effective in inhibiting the secretion of IL-1 in animals, typically IL-1 mediated effects, or A composition that is administered to a mammal in need of suppressing IL-1-mediated inflammation in an amount effective to cause such suppression.
The compounds have relatively few harmful side effects in appropriate doses that suppress IL-1 secretion. The composition of the present invention suppresses the proliferation of T lymphocytes induced by concanvaline-A and the proliferation of B lymphocytes induced by ribopolysaccharide, or the secretion of the immune mediators interleukins 2 and 3. It can be used to suppress IL-1 secretion without significant effects.

Although the hydroxyl group can be in the ortho or para position relative to thiosulfur, a preferred group of compounds used in the present invention are those compounds where the hydroxyl group is para relative to sulfur. These compounds have the formula Corresponding to 4,4'-substituted alkylidene dithio-bis-
(2-tert-butyl-6-alkylphenol). Wherein R ′ and R ″ are hydrogen, methyl, ethyl, propyl, butyl, or pentyl, provided that R ′ and R ″ are not isopropyl, and R ₁ is hydrogen, methyl, or ethyl, and R ₂ is lower alkyl of 1-6 carbon atoms, or keto-substituted lower alkyl of 3-6 carbon atoms, or R ₂ is of the formula Where n is 2, 3 or 4 and R ′, R ″ and R ₁ are as specified above.

Preferred compounds for use in this method are those in which the alkylphenol moieties are all the same, both R groups and R '.
And R ″ are all tert-butyl (t-butyl), all tert-pentyl, Formula I compounds, R ′ and R ″ are both tert-butyl, R ₁ is methyl. And compounds in which R ₂ is methyl or ethyl or propyl. When R ₂ is keto-substituted alkyl, R ₂ is preferably 2-ketoalkyl groups. When R ₂ corresponds to formula II, n is preferably 2 and R ′ and R ″ are preferably tert-butyl. Particularly preferred compounds are both R groups,
A compound corresponding to formula I'where R'and R "are tert-butyl and R ₁ and R ₂ are both methyl. This compound is commonly known as" probucol ". Probucol has been demonstrated to be of low toxicity and to produce no adverse side effects over a wide range of doses.

Some of the compounds are US Patents 3,576,883, 3,78
6,100, 3,862,332 and 3,897,500, all compounds can be made by the methods disclosed in these patents or by analogy to mercaptar and mercaptol known to chemists. Resent,
The compounds are 2 molar equivalents of the appropriate aldehyde or ketone.
It is made by reacting with a molar amount of the appropriate thiophenol. The reaction is conveniently carried out by dispersing thiophenol in an inert organic solvent such as methanol and adding hydrochloric acid (amount of about 0.05 mole HCl per mole thiophenol) as a catalyst. The ketone or aldehyde is then added and the mixture heated to about 30 ° C to boiling temperature for about 1 hour to about 6 hours. When R ₂ is a moiety of formula II or II ′, the aldehyde or ketone reactant is a dialdehyde, ketoaldehyde or diketone and the molar ratio of thiophenol reactant is about thiophenol per mole of aldehyde or ketone reactant. Increased to an amount of 4 mol.

Although the compounds can be administered by conventional routes, oral administration is preferred. The same dosages and routes of administration as those used to reduce cholesterol in alkylidene dithio-bis- (substituted) phenols can generally be used in the practice of the present invention. The appropriate amount used will vary according to factors such as the type of animal being treated, age, weight, condition, and the particular compound used. The optimal dosage for a particular situation can be determined by conventional dosage range determination techniques.

In other words, known suitable levels of compounds, such as US Patent No.
The one described in No. 3,862,332 can be used. The compounds are preferably administered orally in a daily dosage of about 1 mg to about 300 mg of active ingredient per kg of animal body weight. Inhibition of IL-1 release and IL
Results useful in reducing -1-mediated inflammation are animal weight
An oral daily dose of 100 to 200 mg / kg was obtained. The compound is gradually absorbed from the gastrointestinal tract, and for optimal effect,
It is often desirable to continue the administration for several days, or 1-2 weeks or more.

There is some species variation in the response to the compounds used in the methods of the invention. For example, in mice, probucol gives good inhibition of IL-1 release and anti-inflammatory results at a daily dose of 100 mg / kg, whereas in rats, the results obtained in this modest anti-inflammatory test are satisfactory. Was not different. Obtained at a dose of 150 mg / kg in rats
The reduction in IL-1 secretion was lower than that seen at 100 mg / kg in mice. Differences in species-specific responses between rats and mice were also seen in cholesterol reduction by probucol, which showed poor or no response to low doses of probucol in rats, but not in mice, dogs, primates, etc. Other species provide good cholesterol reduction.

The reasons for the differences between species may include factors such as differences in gastric absorption, different rates of compound elimination from the bloodstream, and the like. While it is known that the compounds are relatively non-toxic and have no deleterious side effects, it is relatively preferable to administer the compound in the lowest dosage that produces the desired result.
Where there is species diversity, mammalian species that respond to the compounds are preferred. The species response is suppression of IL-1 release or IL-
It can be determined to test for inhibition of 1-mediated effects. The cholesterol-lowering response of a species to probucol and one of the known cholesterol-lowering compounds is also indicated by the cholesterol-lowering response in that species indicating whether a significant amount of the compound is absorbed or maintained in the circulation.
It may be useful in predicting the responsiveness or non-responsiveness of a species to the IL-1 related effects of compounds.

It is known that the compounds of the present invention and particularly probucol can lower cholesterol, lower LDL and HDL cholesterol, and suppress the oxidation of LDL cholesterol, but for the purpose of the present invention, lower cholesterol in the compounds It is not necessary to administer the compound to animals in need of properties, hypolipidemic and cholesterol oxidation inhibiting properties. Some of the compounds with useful IL-1 release-inhibiting properties have little or no cholesterol-lowering activity. IL-1
Administration of compounds to mammals with non-cholesterolemia mediated by inflammation has beneficial effects in reducing or eliminating inflammation, and non-cholesterol hyperlipidemia to reduce atherosclerotic symptoms. Compounds can be administered to sick animals.

The compounds of the present invention can be formulated in conventional pharmaceutically acceptable carriers to provide convenient unit dosage forms for administration. Generally, known dosage forms and carriers can be used, such as those described in US Pat. No. 3,862,332. A preferred form is 250 mg to 5 mg of active compound, such as the probucol tablets currently used for the treatment of hypercholesterolemia.
It is a tablet containing 00 mg.

Examples The present invention is further illustrated in the following examples.

Example 1 In Vivo Stimulation of IL-1 Release 6-10 week old male mice (CD-1 species, Charles River Laboratories) were used to investigate the effects of compounds on IL-1 release. Each mouse was injected iv with a zymosan suspension containing 2 mg zymosan in 0.5 ml saline for 2 weeks prior to testing. The IL-1 induced acute phase response can be detected 6 hours after intravenous injection of lipopolysaccharide (LPS) at a dose of 100 μg / mouse.
(The LPS used is Salmonella typhimuriu
m) From Re variant. Levi Immunochemical, Hamilton, Missouri. 4.) Intravenous LPS injection into zymosan-treated mice results in IL-1 induced acute phase protein production. One of the acute phase proteins,
Metallothionein causes the removal of zinc from the circulatory system,
This can be measured by atomic absorption as it causes a sharp drop in serum zinc levels.

Daily dose of 100 mg / kg to one group of zymosan-treated Mus musculus
Probucol at a dose of (once daily administration) was orally administered for 14 days before the test. Water was orally administered to the second group and probucol was not administered, and was used as a control group.

On day 14, two subgroups of mice, the saline control subgroup and the probucol saline only subgroup, were tested for serum zinc levels without LPS administration. The other two subgroups, the water / LPS only group and the probucol / LSP group, were administered LPS as described above, the water / LPS subgroup received water and no probucol. Six hours after LPS administration, peripheral blood was collected from individual mice and assayed for zinc. The following table shows the number of mice per group and the result in parts per million zinc (ppm).

Example 2 The procedure of Example 1 was repeated and serum zinc levels were measured again.

Example 3 Treatment of IL-1-Mediated Inflammation Bovine lactobacillus Mycobacter heat-sterilized on the hind paws of Mus musculus
Chronic inflammation was induced in mice by subcutaneous injection of ium butyricum (0.1 ml of 25 mg / ml suspension in mineral oil). In this procedure, one hindpaw was injected and the other was left untreated. The injection produces localized granuloma in about a week and becomes progressively smaller in the next few weeks. The swelling of the granulomatous hindpaw was volumetrically measured and compared to the uninjected hindpaw. This procedure was performed on 4 groups of mice, and swelling measurements were taken at 1, 2, and 5 weeks after the injection of Bovine lactis. Water was administered to one of the groups to serve as a control. Test compounds prednisone, probucol or ibuprofen were administered to the other three groups at 100 mg / day starting from the day of granuloma induction.
It was orally administered at an appropriate dose of kg. Mean swelling of the hindpaw expressed as percent increase in volume (± SEM) compared to contralateral uninjected hindpaw, percent reduction of swelling obtained with test compound, statistically significant difference from control group results (P The level of value) and the number of mice (N) per group are shown in the following table.

Example 4 In Vivo Inhibition of IL-1 Secretion Peritoneal macrophages obtained from CD-1 mice were collected and penicillin 100 units / ml, streptomycin 100 μg / ml,
And Fungizone (GIBCO, Grand Island, NY) RPMI-1640 medium containing 25 μg / ml (GIBCO,
I washed once in Grand Island, NY. The cells were suspended at a cell number of 6 × 10 ⁶ cells / ml and 1 ml aliquots were placed in 6-well flat bottom plates. After culturing at 37 ° C for 1 hour in a humid air chamber containing 5% CO ₂ , non-adhesive cells were removed, and RPMI medium (100 μl of ribopolysaccharide per well
1 ml of medium with or without addition of g) was added. As described in Example 1, LPS stimulates macrophages to release IL-1. Cultivation was continued for 6 hours, after which the culture supernatant was collected and filtered through a 0.22 μm Acrodisc filter (German, Ann Arbor, MI). Liquids were stored at a temperature of -70 ° C until assayed for IL-1 activity.

Mizel et al., J. Immunol. 120: 1497 (1978
IL-1 activity was measured by the C3H / HeJ thymocyte proliferation assay. In this procedure, thymocytes of C3H / HeJ Mus musculus were cultured with macrophage culture medium supernatant in the presence of phytohemagglutinin and pulsed by culture with ³ H-thymidine. Cells were then taken up and radioactivity was measured by liquid scintillation counting. Meisel et al., J.
IL-1 activity was expressed as a unit defined according to Immunol. 120: 1497 (1978).

This procedure pre-incorporates peritoneal macrophages 40, 24, and 16
By oral administration of CD-1 species to mice in time,
The compounds were tested. The dose used was 100 mg / kg.

In one study, peritoneal macrophages from untreated control mice gave 677 ± 59 IL-1 units / ml, whereas the probucol-treated group had only 171 ± 15 IL-1 units / ml assay results. Not given, shows that a 75% inhibition of IL-1 secretion was obtained by oral administration of probucol. In a second study, the mice control group was 15 times that of the probucol-treated group.
Give 960 ± 42 IL-1 unit / ml compared to 3 ± 8 unit / ml, 8
It has decreased by 4%. In the third trial, the control group was 779
± 90 IL-1 unit / ml, but the test for probucol group is
114 ± 13 IL-1 units / ml, indicating that probucol administration results in 86% inhibition of IL-1 secretion. In the fourth test, rats were used as test animals,
The dose of probucol was 150 mg / kg. In this test, the control group received 271 ± 6 units / ml and the probucol-treated rats received 187 ± 15 units / ml, a 31% reduction. The results above are expressed as mean ± standard deviation of the mean. All probucol results differ significantly from controls with p ≦ 0.01.

Example 5 The procedure of Example 4 was repeated using different alkylidene dithio-bis- (substituted) phenol compounds.
The reduction rate of IL-1 secretion obtained by the compounds (rounded to the nearest%) is shown in the following table.

Example 6 Effect on secretion of interleukins-2 and -3 and effect on proliferation of lymphocytes IL-2 is necessary for induction of T lymphocytes and clonal proliferation of T lymphocytes, and IL-3 is granuloma. Is a growth factor. Interleukin-2 and interleukin-
For the effect of probucol on the secretion of 3 (IL-2 and IL-3), Bowlin et al., Cellular Immunol
A similar procedure to that described in ogy 98, p. 341-350 (1986) was used, as in Example 4, using probucol-administered mouse spleen cells and stimulation with concanavalin-A.

In vitro stimulation of spleen cells from mice with probucol administration with concanavalin-A secreted IL-2 and IL-3 to a similar extent as normal spleen cells (from mice without probucol treatment). did. These results indicate that probucol treatment is the immune mediator IL.
2 shows that it does not suppress the induction, synthesis, or secretion of IL-2 and IL-3.

In another study, mitogenic factor ribopolysaccharide (LPS) and concanavalin-A were used to administer spleen cells from normal naive mice and probucol (pre-experiment 40, 24, and
B and T lymphocyte proliferation of spleen cells from mice (100 mg / kg administered for 16 hours) was induced. Growth of spleen cells from probucol-treated mice was not suppressed compared to control normal spleen cell cultures, and a dose of probucol that suppressed IL-1 release suppressed mitogen-induced spleen cell growth. It has no effect.

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Claims (16)

(57) [Claims] (1) Expression [Wherein R is tert-butyl or tert-pentyl, and R '
And R ″ are hydrogen, methyl, ethyl, propyl, butyl, or pentyl, provided that R ′ and R ″ are not isopropyl, R ₁ is hydrogen, methyl, or ethyl, and R ₂ is 1 -Lower alkyl of 6 carbon atoms or keto-substituted lower alkyl of 3-6 carbon atoms,
Or R ₂ A composition for suppressing interleukin-1 release in animals, including humans, comprising an effective amount of a compound corresponding to (wherein n is a moiety corresponding to 2, 3, or 4) . 2. A composition according to claim 1, wherein R, R'and R "all represent tertiary butyl. 3. The compound has the formula [Wherein R is tert-butyl or tert-pentyl, R'and R "are hydrogen, methyl, ethyl, propyl, butyl, or pentyl, provided that R'and R" are not isopropyl; R ₁ is hydrogen, methyl or ethyl,
R ₂ is lower alkyl having 1 to 6 carbon atoms, or 3-
Is a 6 carbon atom keto-substituted lower alkyl, or R ₂ is The composition according to claim 1, which corresponds to (wherein n is 2, 3 or 4). 4. R is tert-butyl, R ₁ is methyl, and R ₂ is 1-.
The composition of claim 2 which is a lower alkyl of 3 carbon atoms. 5. The composition according to claim 4, wherein the compound is probucol. 6. The composition of claim 1, wherein the animal is a mammal, including those who are not hypercholesterolemic. (7) [Wherein R is tert-butyl or tert-pentyl, and R '
And R ″ are hydrogen, methyl, ethyl, propyl, butyl, or pentyl, provided that R ′ and R ″ are not isopropyl, R ₁ is hydrogen, methyl or ethyl,
R ₂ is lower alkyl of 1-6 carbon atoms, or 3-6
Keto-substituted lower alkyl of carbon atoms, or
R ₂ A composition for suppressing an interleukin-1 mediated effect in an animal, comprising an effective amount of a compound corresponding to the formula (n is a moiety corresponding to 2, 3, or 4). 8. A composition according to claim 7, wherein R, R'and R "all represent tertiary butyl. 9. The mammal is a mammal including a person who is not hypercholesterolemia. A composition according to claim 7. (10) a compound represented by the formula: [Wherein R is tert-butyl or tert-pentyl, R'and R "are hydrogen, methyl, ethyl, propyl, butyl, or pentyl, provided that R'and R" are not isopropyl; R ₁ is hydrogen, methyl or ethyl,
R ₂ is lower alkyl having 1 to 6 carbon atoms, or 3-
Is a keto-substituted lower alkyl of 6 carbon atoms]. 11. The composition of claim 10, wherein R ₁ is methyl and R ₂ is lower alkyl of 1-3 carbon atoms. 12. The compound of claim 10, wherein the compound is probucol.
A composition according to claim 1. 13. A composition according to claim 10, wherein R and R'are both tert-butyl, R ₁ is methyl and R ₂ is n-propyl. 14. A composition according to claim 10, wherein R and R'are both tert-butyl, R ₁ is methyl and R ₂ is 2-ketopropyl. 15. The composition of claim 10 wherein R and R'are both tert-butyl, R ₁ is hydrogen and R ₂ is ethyl. 16. The animal is a mammal, including those who are not hypercholesterolemic, and the compound is probucol.
The composition according to claim 10.